Engineering and delivery of therapeutic compositions of freshley isolated cells

ABSTRACT

The present invention relates to the transient modification of cells. In particular embodiments, the cells are immune systems, such as PBMC, PBL, T (CD3+ and/or CD8+) and Natural Killer (NK) cells. The modified cells provide a population of cells that express a genetically engineered chimeric receptor which can be administered to a patient therapeutically. The present invention further relates to methods that deliver mRNA coding for the chimeric receptor to unstimulated resting PBMC, PBL, T (CD3+ and/or CD8+) and NK cells and which delivers the mRNA efficiently to the transfected cells and promotes significant target cell killing.

This application is a continuation application of U.S. patentapplication Ser. No. 16/849,954, filed Apr. 15, 2020, which is acontinuation of U.S. patent application Ser. No. 15/602,536, filed May23, 2017, now U.S. Pat. No. 10,660,917, issued May 26, 2020, which is acontinuation of U.S. patent application Ser. No. 14/834,934, filed Aug.25, 2015, now U.S. Pat. No. 9,669,058, issued Jun. 6, 2017, which is acontinuation of U.S. patent application Ser. No. 13/902,444, filed May24, 2013, now U.S. Pat. No. 9,132,153, issued Sep. 15, 2015, which is acontinuation of U.S. patent application Ser. No. 12/421,352, filed Apr.9, 2009, now U.S. Pat. No. 8,450,112, issued May 28, 2013, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/043,653,filed on Apr. 9, 2008; each of which are hereby incorporated bereference in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of cell biology,cancer biology, and immunology. More particularly, it concerns cellsthat have been engineered by loading them with chemical and biologicalagents and the resultant entities used as therapeutics in the treatmentof multiple indications, including cancer.

2. Description of Related Art

Mononuclear cells, encompassing for example hematopoietic stem cells,mesenchymal stem cells, endothelial progenitor cells, adipose derivedstem cells, and peripheral blood mononuclear cells (PBMC), have beenused in multiple applications for treatment of immune diseases and inregenerative medicine applications (Passweg J and Tyndall A., SeminHematol. 2007 October 44(4):278-85; Le Blanc K and Ringdén O. InternMed. 2007 November 262(5):509-25; Ward et al. Catheter CardiovascInterv. 2007 Dec. 1 70(7):983-98; Mimeault et al., Clin Pharmacol Ther.2007 September 82(3):252-64 Epub 2007 Aug. 1).

Peripheral blood mononuclear cells (PBMC) are comprised of cells ofmyeloid and lymphoid lineages. Myeloid cells, such as monocytes,macrophages, dendritic cells (DC), when loaded with antigens have beendemonstrated to be effective antigen presenting cells for generation oftumor-antigen specific immune responses for treatment of cancer or formodulation of self-antigen specific T cells and regulatory T cells incontrol of autoimmunity (Gilboa E., J Clin Invest. 2007 May117(5):1195-203). Lymphoid cells, such as T cells, NK cells, B cells,lymphoid DC, are effective mediators of immune responses and can befurther harnessed to also present antigen and stimulate naïve and memoryresponses (Hong C and Park S H. Crit Rev Immunol. 2007 27(6):511-25;Martino A and Poccia F, Curr Mol Med. 2007 Nov. 7(7):658-73).

Antigen Presenting Cells (APC) are important sentinels for detecting andpresenting antigens to the immune effector cells. They have beenextensively studied for becoming the effective therapeutic agents.Factors of antigen-loading, process and presentation in the context ofstate of maturity of APC to engage effector cells are major concerns inthe design and development of APC-based immunotherapies and vaccines.Electroloading of tumor antigens, provided in the form of nucleotides(DNA, mRNA) or proteins/lysates or multimeric antigenic formulations,allows for effective uptake and processing of antigens in freshlyisolated cells without requiring efficient maturation of APC antigenuptake mechanisms. Further other chemical and/or biological agents canbe electro-loaded into APC to affect antigen-processing, processedantigen presentation, or immuno-regulatory environment insubject/patient such that the effective biological activity ofelectro-loaded APC is engineered to be superior to that of naïve freshlyisolated APC. Such antigen-loading or antigen-loading combined withenhancement of biological activity for freshly isolated (naïve) APC is aunique attribute of the composition of PBMC thus loaded allowing forrapid formulation and delivery of product to subject/patient. Suchbiological activity otherwise would only be imparted following processesthat require elaborate cell culture, expansion, differentiation,maturation of other manipulation processes that do not lend themselvesto delivery of a therapeutic composition of APC immunotherapy andvaccine products in clinically relevant timeframe for administration tosubject/patient in a hospital/physician's office setting.

NK and T cells are important mediators of viral and tumor immuneresponses. They have been extensively studied for becoming the efficienttherapeutic agents. Factors of efficient and specific target cellkilling, procedure simplicity, cell availability and low graft versushost disease (GvHD) are the major concerns. Chimeric receptor constructshave been described which, when expressed in cells of the immune system,can enhance the immunological specific response to tumor cells andthereby bring clinical benefit to cancer patients. Expanded NK and Tcells expressing a chimeric receptor can overcome HLA-type-relatedinhibition of the expanded NK cell killing and T cell receptor(TCR)-required T cell killing of targeted tumor cells (Imai et al.2004). However, considering the simplicity of using resting NK or Tcells, if the resting NK or T cells, either autologous or allogeneic,could be engineered to have both efficient and specific target cellkilling, it will be the desired for the tumor therapy. Furthermore, ifresting peripheral blood lymphocytes (PBL) or even peripheral bloodmononuclear cells (PBMC) could be engineered to have both efficient andspecific target cell killing, it will be the most desired for the tumortherapy because of procedural simplicity and cell availability. Regularretroviral vectors could not infect resting NK or T cells. Lentiviralvectors have been used to transfect resting peripheral blood lymphocytes(Simmons et al. 2006). Unfortunately, use of viral vectors entailssafety and practical problems for clinical application.

Electroporation is a well recognized method for loading nucleic acidsinto cells to achieve transfection of the loaded cells. The terminologyof electroporation, electrotransfection and electroloading have beeninterchangablly used in the literature with emphasis on general meaningof this technology, the transgene expression and the transference ofmolecules into cytoplasm, respectively. Hereinafter this method oftransfecting cells is referred to as electroloading that is the methodusing electroporation with no transfecting reagent or biologically basedpackaging of the nucleic acid being loaded, such as a viral vector orviral-like particle, relying only on a transient electric field beingapplied to the cell to facilitate loading of the cell. Withinelectroporation, nucleofection is a special one involving a transfectionreagent helping the transferred DNA in the cytoplasm to the nucleus.Nucleofection has been reported to transfect resting T cells and NKcells using plasmid DNA treated with a proprietary nucleofection agent(Maasho et al., 2004). It was also demonstrated that resting T cellnucleofection of chimeric receptor could lead to specific target cellkilling (Finney, et al, 2004). Many reports showed that nucleofection orelectroloading with DNA resulted in cell toxicity to restinghematopoietic cells including lymphocytes, dendritic cells and NK cells(Trompeter et al. 2003; Li et al. 2006; Li et al. 2001; Li et al. 1999;Landi et al., 2007; Van De Parre et al. 2005; Maasho et al. 2004; Abbottet al. 2006). Nucleofected resting NK cells or electrotransfectedresting hematopoietic cells showed good transient viability andefficient transgene expression within a few hours after transfection andlow viability after approximated 28 and 52 hours post-nucleofection andmuch decreased expression of a transgene at these times (Trompeter etal. 2003). Accordingly, this method of transient DNA transfection wouldnot provide for a clinically useful preparation of transiently modifiedresting NK and T cells. Moreover, the transfection efficiency of freshresting NK cells was about half that of growing NK cell lines.

Loading of cells with mRNA brings several advantages, and potentiallycould overcome problems associated with DNA transfection, especially inrespect to resting cells and cells that will be infused into a patient.First, mRNA, especially when loaded by electroloading results in minimalcell toxicity relative to loading with plasmid DNA, and this isespecially true for electroloading of resting cells such as resting NKand peripheral blood mononuclear cells (PBMC) cells. Also, since mRNAneed not enter the cell nucleus to be expressed resting cells readilyexpress loaded mRNA. Further, since mRNA need not be transported to thenucleus, or transcribed or processed it can begin to be translatedessentially immediately following entry into the cell's cytoplasm. Thisallows for rapid expression of the gene coded by the mRNA. Moreover,mRNA does not replicate or modify the heritable genetic material ofcells and mRNA preparations typically contain a single protein codingsequence, which codes for the protein one wishes to have expressed inthe loaded cell. Various studies on mRNA electroloading have beenreported (Landi et al., 2007; Van De Parre et al. 2005; Rabinovich etal. 2006; Zhao et al., 2006).

For a number of medical reasons autologous immunotherapy with restingunstimulated NK, T, PBL, and PBMC and allogeneic immunotherapy forresting unstimulated NK cells can be advantageous for treatment ofcancer. In this context a method that allows removal of cells from thepatient, their treatment outside the body, and their subsequent infusionin to the patient in minimal time, with minimal intervening procedure,and with minimal addition of foreign materials, particularly materialsthat contain replicating genetic information, or are antigenic, isdesired for safety, and reasons of cost and efficiency. A method thatallows modification of these cells without need of extensive cellculture, more specifically without the need for the cells to undergocell division outside the body, comprises loading only of a nucleic acidthat codes for only the therapeutic protein and which is not capable ofreplicating in the cells or modifying the genome of the cell that hasbeen removed from the patient, and which will be returned to thepatient, and which additionally does not involve the use of any otherbiologically or immunologically active components is desired.

SUMMARY OF THE INVENTION

The ability to load freshly prepared unsitmulated resting PBMCs using amethod that only employs nucleic acids (DNA, mRNA, microRNA or RNAi),proteins and small molecules without requiring additional synthetictransfection reagents or viral vectors provides for atransfusion-medicine approach to development of immunotherapy productswhereby PBMCs are removed from a patient or allogenic donor,electroloaded, a process defined herein as electrotransfection, and soonthereafter reinfused into the patient to effect enhanced biologicalactivity in PBMC cell populations leading to enhanced therapeuticeffects for treatment of patients. This therapeutic approach simplifiesthe procedure of obtaining therapeutic compositions of cells thatotherwise could only be obtained following extensive manipulation,including culture, activation, expansion and genetic modification of theexpanded cells. As used herein, a peripheral blood mononuclear cell(PBMC) refers to a blood cell having a nucleus, such as a lymphocyte ormonocyte. An “unstimulated” PBMC refers to a PBMC that has not beenactivated, such as by a cytokine or antigen.

Certain embodiments of the present invention provide methods whereby anucleic acid, such as a DNA or a mRNA, coding for a geneticallyengineered receptor is loaded into NK cells, including resting primaryNK cells, by means of electroloading to provide transiently transfectedNK cells that express the chimeric receptor encoded by the nucleic acid.Also disclosed are methods for transfection of NK cells with nucleicacids, such as DNAs or mRNAs, encoding for more than one chimericreceptor or a combination of a chimeric receptor with other chemicaland/or biological agents. In some embodiments, the present inventionalso provides for engineering NK cells by loading with a nucleic acid,such as a DNA or a mRNA, encoding for a chimeric receptor which can beused as an immunotherapeutic cell therapy for the treatment of cancer ordisease of the immune system.

In one embodiment, the present invention provides a method fortransiently modifying a resting primary peripheral blood mononuclearcell (PBMC) to expresses a chimeric receptor on its surface comprising:isolating resting primary PBMCs; and electroloading the PBMCs with anucleic acid, such as a DNA or an mRNA, encoding a chimeric receptor,whereby the electroloaded PBMCs express the chimeric receptor on itssurface. In certain aspects of the invention the PBMCs are monocytes. Insome aspects of the invention, the PBMCs are peripheral bloodlymphocytes (PBLs). In some embodiments, the lymphocytes are naturalkiller (NK) cells, CD3+ T cells, and/or CD8+ T cells. Resting PBMCs arePBMCs directly collected from peripheral blood or are thawed PBMCs thatwere frozen directly after collection from peripheral blood. RestingPBMCs may be cultured for a short time (e.g., less than 2 days) with orwithout specific stimulation of cytokines or ligands to stimulate cellactivation for cell number expansion.

In one embodiment, the present invention provides a method fortransiently modifying a natural killer (NK) cell to expresses a chimericreceptor on its surface comprising: isolating an NK cell; andelectroloading the NK cell with a nucleic acid, such as a DNA or anmRNA, encoding a chimeric receptor, whereby the electroloaded NK cellexpresses the chimeric receptor on its surface.

The NK cell may be a resting NK cell or a growing NK cell line. RestingNK cells are NK cells directly collected from peripheral blood or arethawed NK cells that were frozen directly after collection fromperipheral blood. Resting NK cells may be cultured for a short time(e.g., less than 2 days) with or without specific stimulation ofcytokines or ligands to stimulate cell activation for cell numberexpansion. Growing NK cells are cells that have undergone cellstimulation/activation with a cytokine and ligand to activate cells toexpand in cell number.

An “isolated” NK cell or “isolating” an NK cell refers to separating NKcells from non-NK cells such as red blood cells, monocytes, T cells, andB cells. A variety of methods are known for the isolation of NK cellsand kits are commercially available for this purpose. When NK cells arebeing isolated from whole blood, it may be desirable to first separate(by centrifugation, for example) the red blood cells from immune-systemcells, and then to further separate the NK cells from other types ofimmune-system cells. One approach for separating NK cells from othercells is based on the expression of different surface markers ondifferent cell types. For example, one can select for NK cells withantibodies that bind CD56 or CD16, which are expressed on the surface ofNK cells, for positive selection. Thus, in one aspect of the invention,isolating NK cells comprises separation of CD56+ cells from CD56− cells.In another aspect of the invention, isolating NK cells comprisesseparation of CD16+ cells from CD16− cells. In a further aspect of theinvention, isolating NK cells comprises separation of CD56+ and CD16+cells from CD56− and CD16− cells. Antibodies used for isolating NK cellswill generally be attached to a solid support and/or magnetic particles(e.g., magnetic beads) to facilitate the separation of the capturedcells from those cells that were not bound by the antibody. Isolation ofNK cells may also comprise depletion (i.e., negative selection) ofnon-NK cells from the sample by binding surface markers, such as CD14,CD3, and/or CD19, which are not expressed on the surface of NK cells.Thus, in one aspect of the invention, isolating NK cells comprisesdepleting CD14+, CD3+, and/or CD19+ cells from the sample.

An “isolated” T cell or “isolating” a T cell refers to separating Tcells from non-T cells such as red blood cells, monocytes, NK cells, andB cells. A variety of methods are known for the isolation of T cells andkits are commercially available for this purpose. When T cells are beingisolated from whole blood, it may be desirable to first separate (bycentrifugation, for example) the red blood cells from immune-systemcells, and then to further separate the T cells from other types ofimmune-system cells. One approach for separating T cells from othercells is based on the expression of different surface markers ondifferent cell types. For example, one can select for T cells withantibodies that bind CD3, which is expressed on the surface of T cells,for positive selection. Isolation of T cells may also comprise depletion(i.e., negative selection) of non-T cells from the sample by bindingsurface markers, such as CD56 and CD16, which are not expressed on thesurface of T cells. PBLs, may be isolated from other non-PBL PBMCs byculturing the PBMCs in a container (e.g., flask) and removing the cellsthat attach to the surface of the container after about 1-2 hours.

The purity of isolated cells may be determined by, for example,fluorescence-activated cell sorting (FACS). In one embodiment of theinvention, isolating NK cells or T cells comprises isolating peripheralblood lymphocytes (PBLs) from other cells, such as red blood cells andmonocytes. In certain aspects, the PBLs comprise at least 70%, 80%, 90%,95%, 97%, 99%, or 99.5% of the cells in a composition. In anotherembodiment of the invention, isolating NK cells comprises isolating theNK cells from all other types of cells, including other PBLs. In certainaspects, the NK cells comprise at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 97%, 99%, or 99.5% of the cells in a composition. Inanother embodiment of the invention, isolating T cells comprisesisolating the T cells from all other types of cells, including otherPBLs. In certain aspects, the T cells comprise at least 90%, 95%, 97%,99%, or 99.5% of the cells in a composition.

The terms “transient transfection” and “transiently modifying” refer tothe introduction of a nucleic acid molecule into a cell using atransfection process that does not usually result in the introducednucleic acid molecule being inserted into the nuclear genome, theintroduced nucleic acid molecule is, therefore, lost as the cellsundergo mitosis. In contrast, “stable transfection” refers to atransfection process in which cells that have integrated the introducednucleic acid molecule into their genome are selected. In this way, thestably transfected nucleic acid remains in the genome of the cell andits daughter cell after mitosis. The term “transiently expressing”refers to the transient expression of a nucleic acid molecule in atransiently transfected cell.

In one embodiment, the present invention provides a method of treating ahyperproliferative disease in a subject comprising: obtaining isolatedresting primary peripheral blood mononuclear cells (PBMCs);electroloading the PBMCs with a nucleic acid, such as a DNA or an mRNA,coding for a chimeric receptor, whereby the electro-loaded PBMCs expressthe chimeric receptor on its surface; and administering the transfectedPBMCs to the subject to treat the hyperproliferative disease in thesubject. In certain embodiments, the PBMCs are obtained from a donorother than the subject being treated. In other embodiments, the PBMCsare obtained from the subject with the hyperproliferative disease.

In one embodiment, the present invention provides a method of treating ahyperproliferative disease in a subject comprising: obtaining isolatedNK cells from a subject with a hyperproliferative disease or from adonor; electroloading the NK cells with a nucleic acid, such as a DNA oran mRNA, coding for a chimeric receptor, whereby the electroloaded NKcells express the chimeric receptor on their surfaces; and administeringthe transfected NK cells to the subject to treat the hyperproliferativedisease in the subject. In certain embodiments, the NK cells are freshlycollected primary NK cells. In one aspect, the freshly collected primaryNK cells are isolated and electro-loaded immediately after they areobtained from the subject.

In some embodiments, the freshly collected primary PBMCs are collected,isolated, and transfected within about 0.5 to 3 hours, 0.5 to 2 hours,or 0.5 to 1 hour. In some embodiments, the freshly collected primaryPBMCs are frozen immediately after being collected from patient. ThePBMCs may be frozen in peripheral blood or they may be isolated and thenfrozen or they may be isolated, transfected and then frozen. Thus, incertain aspects of the invention, fresh primary PBMCs may be thawedcells that were frozen immediately after collection from a patient/donoror immediately after isolation following collection. In someembodiments, the transfected cells are administered to the patientwithin about 1 to 48 hours, 1 to 24 hours, 1 to 15 hours, 1 to 10 hours,or 1 to 5 hours from the time the cells were originally obtained fromthe patient or donor. In some aspects, freshly collected cells are cellsthat have been collected from a subject but have not undergone celldivision outside of the subject; thus, administering freshly collectedcells to a subject would refer to administering cells that have notundergone cell division outside of a subject.

In certain embodiments, the subject is a human. In one embodiment, thehyperproliferative disease is cancer. It is contemplated that any typeof cancer can be treated with the methods and compositions disclosedherein, including, for example, breast cancer, lung cancer, prostatecancer, ovarian cancer, brain cancer, liver cancer, cervical cancer,colon cancer, renal cancer, skin cancer, head & neck cancer, bonecancer, esophageal cancer, bladder cancer, uterine cancer, lymphaticcancer, stomach cancer, pancreatic cancer, testicular cancer, orleukemia. The leukemia may be, for example, acute lymphocytic leukemia(ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia(CLL), chronic myelogenous leukemia (CML), or mantle cell lymphoma(MCL).

The transfected cells may be administered to the subject by methods wellknown to those of skill in the art. For example, the transfected cellsmay be administered by intravenous injection, intraarterial injection,intralymphatic injection, intramuscular injection, intratumoralinjection, or subcutaneous injection. It is also contemplated that thetransfected cells may be administered intraperitoneally. The transfectedcells may be administered to the subject at or near a tumor in thesubject, or to a site from which a tumor has been surgically removedfrom the subject. However, it is not necessary that the transfectedcells be administered at the tumor site to achieve a therapeutic effect.Thus, in certain embodiments the transfected cells may be administeredat a site distant from the tumor site. A medical practitioner will beable to determine a suitable administration route for a particularsubject based, in part, on the type and location of thehyperproliferative disease. The transfected cells may be administeredlocally to a disease site, regionally to a disease site, orsystemically. In one embodiment, the cells are administered byintravenous injection or intralymphatic injection. In anotherembodiment, the transfected cells are administered locally to a tumorsite, such as by intratumoral injection. In some embodiments, thetransfected cells are administered back in to the patient in less than48 hours, less than 24 hours, or less than 12 hours from the time fromwhen the peripheral blood is collected from the donor. In certainaspects of the invention, the transfected cells are administered back into the patient within about 1 to 48 hours, 1 to 24 hours, 1 to 15 hours,1 to 12 hours, 1 to 10 hours, or 1 to 5 hours from the time the NK cellswere originally obtained from the donor. The donor and the subject beingtreated may be the same person or different people. Thus, in someembodiments the cells are autologous to the subject; and in otherembodiments, the cells are allogenic to the subject.

The chimeric receptor will generally be selected based on the cell beingtargeted for killing. Thus, in one embodiment of the invention, thechimeric receptor is a chimeric receptor that binds a tumor antigen.CD19 is expressed on B-lineage cells. Accordingly, to kill leukemic Bcells an anti-CD19 chimeric receptor could be expressed on the surfaceof a PBMC, such as a NK cell, which would enhance interaction betweenthe modified NK cells and B cells. Thus, in one embodiment of theinvention, the chimeric receptor is an anti-CD19 chimeric receptor. Inone aspect, the anti-CD19 chimeric receptor is an anti-CD19BBz encodinga single chain antibody conjugated with the 4-1 BB intercellular domainand the CD3ζ domain. In certain embodiments, the chimeric receptor is ananti-CD20, anti-FBP, anti-TAG-72, anti-CEA, anti-carboxyanhydrase IX,nati-CD171, anti-IL-13 receptor, anti-G(D)2, anti-PSMA, anti-mesothelin,anti-Lewis-Y, or anti-CD30 chimeric receptor. CARs directed to theseantigens may be used to treat the diseases associated with the cellsthat express these antigens. For example, these antigens have beenassociated with at least the following tumors: CD-19 (leukemia), FBP(ovarian), TAG-72 (colorectal), CEA (colorectal, breast, gastric),carboxyanhydrase IX (renal), CD171 (neuroblastoma), IL-13 receptor(glioblastoma), G(D)2 (neuroblastoma), PSMA (prostate), mesothelin(pancreatic), Lewis-Y (myeloma), or CD30 (cutaneous lymphoma). Incertain aspects of the invention, the chimeric receptor does not containan intracellular domain. In certain embodiments, the chimeric receptordoes not contain a CD28 intracellular domain.

In another embodiment, the present invention provides a compositioncomprising: an electroloaded PBMC transiently expressing transgeneencoded by a nucleic acid, such as a DNA or an mRNA, coding for achimeric receptor, whereby the chimeric receptor is expressed on thesurface of the electro-loaded PBMC; and a pharmaceutically acceptablecarrier. In one aspect of the invention, the PBMC is a resting PBMC. Inanother aspect of the invention, the composition is frozen. The chimericreceptor may be, for example, an anti-CD19 chimeric receptor. In someembodiments, the composition does not contain a DNA, such as a DNAplasmid, encoding the chimeric receptor. In certain embodiments, thecomposition is free or substantially free of viral vectors andviral-like particles.

In one embodiment, the present invention provides a compositioncomprising: an electrotransfected NK cell transiently expressingtransgene encoded by a mRNA coding for a chimeric receptor, whereby thechimeric receptor is expressed on the surface of the electrotransfectedNK cell; and a pharmaceutically acceptable carrier. In one aspect of theinvention, the NK cell is a resting NK cell. In another aspect of theinvention, the composition is frozen. The chimeric receptor may be, forexample, an anti-CD19 chimeric receptor. In some embodiments, thecomposition does not contain a DNA, such as a DNA plasmid, encoding thechimeric receptor. In certain embodiments, the composition is free orsubstantially free of non-NK cells. In certain aspects, at least 60%,80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5%, or 99.9% of the cells in thecomposition are NK cells.

The present invention also provides for loading antigens into PBMCs, andin particular in to antigen presenting cells (APCs), or for loading saidantigens along with other chemical or biological agents that enhanceeffectiveness of antigen processing, antigen presentation, celltrafficking and localization, and control of immunoregulatoryenvironment in a subject/patient, to facilitate use of freshly isolated(naïve) and electro-loaded PBMCs as therapeutic compositions and methodsfor treatment of cancer and immune diseases.

Those of skill in the art are familiar with methods of electroporation.The electroporation may be, for example, flow electroporation or staticelectroporation. In one embodiment, the method of transfecting thecancer cells comprises use of an electroporation device as described inU.S. patent application Ser. No. 10/225,446, incorporated herein byreference. Methods and devices for electroporation are also describedin, for example, published PCT Application Nos. WO 03/018751 and WO2004/031353; U.S. patent application Ser. Nos. 10/781,440, 10/080,272,and 10/675,592; and U.S. Pat. Nos. 5,720,921, 6,074,605, 6,773,669,6,090,617, 6,485,961, 6,617,154, 5,612,207, all of which areincorporated by reference.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

Any embodiment of any of the present methods, devices, and systems mayconsist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1E. Macromolecule loading/transfection in expanded NK cells.FIG. 1A shows that FITC-dextran (500 k MW) could be efficiently loadedinto expanded NK cells. FIG. 1B shows that FITC-siRNA (21-mer) could beefficiently loaded into expanded NK cells. FIG. 1C shows that DNAplasmid encoding eGFP driven by CMV promoter could be transfected into6-day expanded NK cells. FIG. 1D shows that mRNA encoding eGFP could beefficiently transfected into expanded NK cells with no significantviability lose. FIG. 1E shows proliferation of eGFP-mRNA transfectedcells.

FIGS. 2A-2B. Transfection of mRNA encoding anti-CD19 chimeric receptorin expanded NK cells. FIG. 2A shows that 65% of viable cells couldexpress anti-CD19 chimeric receptor. FIG. 2B shows that the anti-CD19chimeric receptor expression was mRNA concentration dependent, and couldexpress up to 4-5 days.

FIGS. 3A-3D. Resting NK cells could be efficiently transfected withmRNA. FIG. 3A shows NK cell phenotype after Miltenyi bead isolation.FIG. 3B shows that resting NK cells could be efficiently transfectedwith mRNA encoding eGFP or anti-CD19 chimeric receptor. FIG. 3C showsthe expression duration of the anti-CD19 chimeric receptor in resting NKcells. FIG. 3D shows a summary of viability and expression level ofresting NK cells from two donors.

FIGS. 4A-4B. Effect of CD3+ cells on OP-1 killing by transfected NKcells. FIG. 4A shows that CD3+ cells could be efficiently depleted withDynal bead from 28% to 0.4%. FIG. 4B shows that either depletion or nodepletion of CD3+ cells from expanded NK cells did not affect the NKcell killing of OP-1 cells.

FIGS. 5A-5C. Specificity of NK cell killing. FIG. 5A shows that propertransfection resulted in expression of the transgene. FIG. 5B showstypical FACS data that GFP-expressed NK cells did not kill CD19-PE+ OP-1cells (3rd panels from left). Only anti-CD19 chimeric receptor-expressedNK cells killed OP-1 significantly (4th panels from left). FIG. 5C showsa summary of anti-CD19 chimeric receptor-specific OP-1 killing.Electroporation alone (calcein-AM method) and GFP-transfected NK cells(antibody staining) did not kill OP-1. Anti-CD19 chimericreceptor-expressed NK cells significantly killed OP-1.

FIGS. 6A-6D. Duration of killing by expanded NK cells transfected withanti-CD19 chimeric receptor. Killing was analyzed by calcein-AM method,set up at 3 h (FIG. 6A), 1 day (FIG. 6B), 2 days (FIG. 6C), and 3 days(FIG. 6D) post transfection, and analyzed after 4 hours or 1 daykilling.

FIGS. 7A-7B. OP-1 cell killing by NK cells from two different donors.FIG. 7A shows that NK cells expanded from two different donors led tosimilar and significant OP-1 cell killing. FIG. 7B shows that resting NKcells from two different donors led to similar and significant OP-1 cellkilling.

FIGS. 8A-8B. Allogeneic primary B-CLL cell killing by anti-CD19 chimericreceptor-expressed NK cells. FIG. 8A shows significantly higher cellkilling of B-CLL cells by expanded NK cells with anti-CD19 chimericreceptor expression than that by naïve expanded NK cells. B-CLL cellswere from two donors. FIG. 8B shows that B-CLL cells could bespecifically killed by resting NK cells with anti-CD19 chimeric receptorexpression. The killing was efficient for at least 2 days aftertransfection.

FIGS. 9A-9D. DNA uptake is toxic to resting PBL. Resting PBL waselectroporated in the presence of plasmid DNA encoding for eGFP underCMV promoter, mRNA encoding for the eGFP and macromolecule FITC-dextran(500 kD). The viability and the expression level were monitored bytrypan blue exclusion and flow cytometry analysis for up to 7 days posttransfection. FIG. 9A shows typical FACS analysis data. FIG. 9B showsdependence of viability on time post transfection. FIG. 9C showsdependence of viable cell numbers on time post transfection. FIG. 9Dshows dependence of expression on time post transfection.

FIGS. 10A-10B. DNA uptake resulted in enhanced apoptosis in resting PBL.Resting PBL was transfected with 200 ug/ml of plasmid DNA encoding forDsRed under CMV promoter and analyzed 1d post transfection. Thetransfected cells were labeled with apoptosis indicator FITC-VAD-FMK(Promega, Madison, Wis.) following the product instruction. Apoptosisand transgene expression of the transfected cells were analyzed by FACS(FIG. 10A). The transfected cells without EP, DNA or caspase inhibitor(Enzyme System Product, Livermore, Calif.) (−E−D−I), with EP but withoutDNA or caspase inhibitor (+E−D−I), with EP and caspase inhibitor butwithout DNA (+E−D+I), with EP and DNA but without caspase inhibitor(+E+D−I), and with EP, DNA and caspase inhibitor (+E+D+I) were analyzedwith Cell Death Detection ELISAPLUS kit (Roche, Indianapolis, Ind.)(FIG. 10B). Caspase inhibitor could only slow the cell death of theDNA-transfected resting PBL, not stop it (data not shown).

FIG. 11A-11E. αCD19 chimeric expression in resting PBMC (FIG. 11A),resting NK cells (FIG. 11B), resting PBL (FIG. 11C), resting PBMC fromCLL patient (FIG. 11D), resting T cells from CLL cells (FIG. 11E). −E or+E denotes the samples with or without transfection. −CD19 or +CD19represents the samples with or without addition of 5×10⁴/ml (about 3% ofthe total cells in culture) autologous CD19+ CLL cells. 100 IU/ml hIL-2was added in the cell culture.

FIG. 12A-12D. Characteristics of transfected resting PBL with αCD19chimeric receptor. FIG. 12A shows dependence of viability of transfectedPBL on time post transfection. FIG. 12B shows dependence of viable cellrecovery on time post transfection. FIG. 12C shows expansion oftransfected PBL analyzed with CFSE. FIG. 12D shows expansion of CD3+cells analyzed with CFSE.

FIG. 13A-13D. Specificity of allogeneic target cell/cell line killing byαCD19 chimeric receptor-transfected resting PBL. FIG. 13A shows typicalFACS analysis result of OP-1 cell line killing. FIG. 13B shows specificOP-1 killing by 2 donors of transfected PBL. FIG. 13C shows non-specificK562 cell killing by transfected PBL. FIG. 13D shows specific CLL cellkilling.

FIG. 14A-14E. Specific autologous B (FIG. 14 A-C) or purified-CD19+ CLL(FIGS. 14 D and E) cell killing by Resting NK cells (1d posttransfection) (FIG. 14A); resting PBMC (3d post transfection) (FIG.14B); Resting PBL (FIG. 14C, 1d post transfection); resting PBMC fromCLL patient (FIG. 14D, 2d post transfection) and resting CD3+ cells fromCLL patient (FIG. 14E, 3d post transfection) after transfection withαCD19 chimeric receptor.

FIG. 15A-15D. Duration of specific killing of autologous B cells bytransfected resting PBL with αCD19 chimeric receptor. The four-hourkilling assay was performed at 1d post transfection (FIG. 15A), 2d posttransfection (FIG. 15B), 3d post transfection (FIG. 15C), and 7d posttransfection (FIG. 15D).

FIG. 16. HS-Sultan lymphoblastic cell killing by CAR-transfected PBMCsin vitro.

FIG. 17. HS-Sultan lymphoblastic cell killing by CAR-transfected PBMCsin vivo. HS-Sultan cells (1e6 cells) mixed with CAR-transfected PBMCs(0.7e6, 2.3e6, 6.7e6 and 20e6 respectively) were injected subcutaneouslyinto beige SCID mice. The tumor volume was measured at indicated timepoints.

FIG. 18. Cytokine-induced NK cells (LAK) transfected with mRNA encodinganti-CD19-BBz exhibit greater cytotoxicity against HS-Sultan cells thanstimulated LAK that were not transfected with mRNA encodinganti-CD19-BBz.

FIG. 19. The effect of intracellular domains on CAR expression inexpanded T cells. The percent CAR expression is shown on the y axis. Thetime post transfection is shown on the x axis.

FIG. 20. The effect of intracellular domains on CAR expression inexpanded T cells. The MFI is shown on the y axis. The time posttransfection is shown on the x axis.

FIG. 21. K562 killing by expanded T cells transfected with CARs linkedto different intracellular domains.

FIG. 22. The CD28 intracellular domain decreases expression of CARsfaster in T cells than in unstimulated resting PBMCs.

FIG. 23. Unstimulated resting PBMCs transfected with the ss1-28z CARmaintained K562+ cell killing at four days post transfection.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides methods and compositions for theprevention and treatment of diseases, such as cancer and otherhyperproliferative diseases. In certain embodiments, present inventionprovides methods for the preparation of transiently modified NK, T, PBL,and/or PBMC cells that provide previously unattained levels of cellviability following transfection, expression of a chimeric receptor thatenhances specific anti-tumor activity by the modified cells, convenienceand clinical applicability in autologous and allogeneicimmunotherapeutic regimen, and improved precision in the transientmodification, and safety in terms of risk of engineering of transfectedcells. The methods are applicable to a wide range of chimeric receptorconstructs and therapeutic proteins.

The ability to load freshly obtained resting unstimulated cells, fromsources such as, for example peripheral blood, bone marrow, fat or otherorgan/tissue sources, using a method that employs transient energydelivery to facilitate transfer of chemical and/or biological agents,such as for example nucleotides (DNA, mRNA, microRNA or RNAi), proteinsand small molecules, across a lipid bi-layer to affect the biologicalactivity of desired cells within the freshly isolated cell population,wherein the affected biological activity is enhanced compared to thefreshly isolated (non-loaded) cell compositions, and wherein the saidcomposition of engineered cells can be safely delivered within aclinically relevant time-frame to a patient within a hospital and/orphysician's office setting without requiring extensive needs forculture, expansion, differentiation or manipulation of cells, providesfor unique therapeutic compositions of cells, in the context of atransfusion-medicine like approach to the development and delivery ofnovel therapeutic products, as effective treatment for multiple immunediseases.

Specifically, an approach to development of immunotherapy productswhereby unstimulated mononuclear cells obtained from peripheral bloodare obtained from a patient, loaded with relevant chemical or biologicalagents using transient delivery of energy, such as electrical, light,sound, heat, waves, and chemical and/or biological mediation, to affectthe biological activity of freshly isolated mononuclear cells, and soonthereafter reinfused into the patient to effect enhanced biologicalactivity in specific mononuclear cell populations contained with thefreshly isolated cells leading to enhanced therapeutic effects fortreatment of patients. This therapeutic approach may provide analternative to the use of purified, isolated, or enriched cells, thatneed to be expanded/activated and transformed to impact their biologicalactivity (potency) and thus may be preferred in multiple situationsrequiring medical interventions.

Mononuclear cells obtained from multiple sources (peripheral blood, bonemarrow aspirates, lipo-aspirates, tissue-specific perfusates/isolates)can be effectively loaded with chemical and/or biological agents in acontrolled manner using electrical energy, thereafter referred to aselectroloading, to obtain desired level and duration of modulation ofmolecular pathways. Controlled intervention of molecular pathwaysprovides means for affecting biological activity of cells whenadministered back to subject/patient, thus enhancing the ability tomitigate potency and efficacy that is otherwise not provided for in theadministration of unmodified, freshly isolated cells.

A. NATURAL KILLER CELLS

In certain embodiments, the present invention employs geneticallymodified natural killer cells in the treatment of hyperproliferativediseases. Natural killer cells (NK cells) are a type of cytotoxiclymphocyte. NK cells are activated in response to interferons ormacrophage-derived cytokines, and they play a major role in therejection of tumors and cells infected by viruses. NK cells kill cancercells and virally infected cells by releasing small cytoplasmic granulescalled perforin and granzyme that cause the target cell to die.

NK cells are characterized by their lack of the T cell receptor (CD3)and their expression of CD56 on their surface. Accordingly, thesecharacteristics may be used to separate NK cells from other cell types.In contrast to cytotoxic T lymphocytes (CTL), NK cells do not requireantigen activation and are not MHC restricted.

Cancer cells may evade killing by NK cells because self HLA molecules onthe cancer cells can bind to the killer immunoglobulin-like receptors(KIRs) and inhibit the NK cell killing. The present invention providesmethods and compositions that overcome this inhibition and promotes NKcell killing of cancer cells.

B. T CELLS

In some embodiments, the present invention employs genetically modifiedT cells in the treatment of hyperproliferative diseases. T cells play arole in cell-mediated immunity. One way in which T cells can bedistinguished from other lymphocytes, such as B cells and NK cells, isby the presence on their cell surface of the T cell receptor (TCR).Activation of CD8+ T cells and CD4+ T cells occurs through theengagement of both the T cell receptor and CD28 on the T cell by themajor histocompatibility complex (MHC) peptide and B7 family members onan antigen presenting cell (APC). Engagement of the T cell receptor forantigen (TCR) in the absence of CD28 costimulation can result in along-term hyporesponsive state termed clonal anergy (Schwartz, 2003).Anergic T cells show defective IL-2 production and proliferation uponrestimulation via the TCR and CD28, and produce other cytokines atreduced levels. Anergy may represent one mechanism of peripheraltolerance (Ramsdell et al., 1989), and has been reported to occur in thesetting of non-productive anti-tumor immunity in vivo(Staveley-O'Carroll et al., 1998).

C. CHIMERIC RECEPTORS

Chimeric receptors generally comprise an extracellular antibody tospecific antigen on the target cell surface and anactivation/stimulation domain in the cytoplasm. chimeric receptorexpression in NK, T, PBL, or PBMC cells directly links the NK, T, PBL,or PBMC cells to target cells and consequently allow NK or T cells tokill the target cells. Under this mechanism, the target cell killing canavoid the HLA-type—related NK cell killing inhibition and T cellreceptor (TCR)—requirement for T cell-induced target cell killing. Inone embodiment of the invention, the chimeric receptor is an anti-CD19chimeric receptor comprising a single chain antibody conjugated with the4-1 BB intracellular domain and the CD3ζ domain. Chimeric receptormolecules are described in US 2004/0038886, which is incorporated hereinby reference.

D. HYPERPROLIFERATIVE DISEASES

The invention may be used in the treatment and prevention ofhyperproliferative diseases including, but not limited to, cancer. Ahyperproliferative disease is any disease or condition which has, aspart of its pathology, an abnormal increase in cell number. Included insuch diseases are benign conditions such as benign prostatic hypertrophyand ovarian cysts. Also included are premalignant lesions, such assquamous hyperplasia. At the other end of the spectrum ofhyperproliferative diseases are cancers. A hyperproliferative diseasecan involve cells of any cell type. The hyperproliferative disease mayor may not be associated with an increase in size of individual cellscompared to normal cells.

Another type of hyperproliferative disease is a hyperproliferativelesion, a lesion characterized by an abnormal increase in the number ofcells. This increase in the number of cells may or may not be associatedwith an increase in size of the lesion. Examples of hyperproliferativelesions that are contemplated for treatment include benign tumors andpremalignant lesions. Examples include, but are not limited to, squamouscell hyperplastic lesions, premalignant epithelial lesions, psoriaticlesions, cutaneous warts, periungual warts, anogenital warts,epidermdysplasia verruciformis, intraepithelial neoplastic lesions,focal epithelial hyperplasia, conjunctival papilloma, conjunctivalcarcinoma, or squamous carcinoma lesion. The lesion can involve cells ofany cell type. Examples include keratinocytes, epithelial cells, skincells, and mucosal cells.

E. CANCER

The present invention provides methods and compositions for thetreatment and prevention of cancer. Cancer is one of the leading causesof death, being responsible for approximately 526,000 deaths in theUnited States each year. The term “cancer” as used herein is defined asa tissue of uncontrolled growth or proliferation of cells, such as atumor.

Cancer develops through the accumulation of genetic alterations (Fearonand Vogelstein, 1990) and gains a growth advantage over normalsurrounding cells. The genetic transformation of normal cells toneoplastic cells occurs through a series of progressive steps. Geneticprogression models have been studied in some cancers, such as head andneck cancer (Califano et al., 1996). Treatment and prevention of anytype of cancer is contemplated by the present invention. The presentinvention also contemplates methods of prevention of cancer in a subjectwith a history of cancer. Examples of cancers have been listed above.

F. ELECTROPORATION

Certain embodiments involve the use of electroporation to facilitate theentry of one or more nucleic acid molecules into cells of the immunesystem, such as natural killer (NK) cells.

As used herein, “electroporation” refers to application of an electricalcurrent or electrical field to a cell to facilitate entry of a nucleicacid molecule into the cell. One of skill in the art would understandthat any method and technique of electroporation is contemplated by thepresent invention. However, in certain embodiments of the invention,electroporation may be carried out as described in U.S. patentapplication Ser. No. 10/225,446, filed Aug. 21, 2002, the entiredisclosure of which is specifically incorporated herein by reference.

In other embodiments of the invention, electroloading may be carried outas described in U.S. Pat. No. 5,612,207 (specifically incorporatedherein by reference), U.S. Pat. No. 5,720,921 (specifically incorporatedherein by reference), U.S. Pat. No. 6,074,605 (specifically incorporatedherein by reference); U.S. Pat. No. 6,090,617 (specifically incorporatedherein by reference); and U.S. Pat. No. 6,485,961 (specificallyincorporated herein by reference).

Other methods and devices for electroloading that may be used in thecontext of the present invention are also described in, for example,published PCT Application Nos. WO 03/018751 and WO 2004/031353; U.S.patent application Ser. Nos. 10/781,440, 10/080,272, and 10/675,592; andU.S. Pat. Nos. 6,773,669, 6,090,617, 6,617,154, all of which areincorporated by reference.

G. PHARMACEUTICAL PREPARATIONS

1. Formulations

Pharmaceutical preparations of transfected cells for administration to asubject are contemplated by the present invention. One of ordinary skillin the art would be familiar with techniques for administering cells toa subject. Furthermore, one of ordinary skill in the art would befamiliar with techniques and pharmaceutical reagents necessary forpreparation of these cell prior to administration to a subject.

In certain embodiments of the present invention, the pharmaceuticalpreparation will be an aqueous composition that includes the transfectedcells that have been modified to express genetically engineeredreceptor. In certain embodiments, the transfected cell is prepared usingcells that have been obtained from the subject (i.e., autologous cells).

Pharmaceutical compositions of the present invention comprise aneffective amount of a solution of the transfected cells in apharmaceutically acceptable carrier or aqueous medium. As used herein,“pharmaceutical preparation” or “pharmaceutical composition” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the transfected cancer cells, its use in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions. For humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by the FDA Center forBiologics.

The transfected cancer cells may be formulated for administration by anyknown route, such as by subcutaneous injection, intramuscular injection,intravascular injection, intratumoral injection, or application by anyother route. A person of ordinary skill in the art would be familiarwith techniques for generating sterile solutions for injection orapplication by any other route. Determination of the number of cells tobe administered will be made by one of skill in the art, and will inpart be dependent on the extent and severity of cancer, and whether thetransfected cells are being administered for treatment of existingcancer or prevention of cancer. The preparation of the pharmaceuticalcomposition containing the transfected cells will be known to those ofskill in the art in light of the present disclosure. The transfectedcells may be administered with other agents that are part of thetherapeutic regiment of the subject, such as other immunotherapy orchemotherapy. In some embodiments, about 1e7, 1e8, 1e9, or 1e10, or anyrange derivable therein, of transfected cells are administered per dose.In certain aspects, multiple doses may be administered over a period ofdays, weeks, months, or year. A subject may receive, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20doses.

H. EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Electroloading of NK Cells with a DNA Plasmid Encoding aMarker Gene

NK cells were transfected using electroporation with a DNA plasmidcarrying an eGFP marker gene. One day after transfection, the viable andtransfected NK cells were assayed and found to be about 50% and 30%respectively, as shown in FIG. 1C. When normalized to untransfectedcontrol cells, the viability of transfected NK cells using this plasmidDNA was about 60%. Approximately 50% of the viable NK cells expressedthe eGFP marker gene. No significant change in phenotype was observed.Even though NK cells could be transfected well 1 day post transfection,the transfected NK cells lost their proliferation ability and viabilitybecause of DNA-uptake mediated cytotoxicity. DNA transfection of NKcells, therefore, would not result in meaningful clinical application.

Example 2 Electroloading of Expanded NK Cells with mRNA Coding for aMarker Gene

As shown in FIG. 1D, mRNA coding for eGFP was electrotransfected intoexpanded NK cells. About 80% of the viable cells expressed GFP. At thesame time, the viability of NK cells was 67% in the untransfectedcontrol and 60% in the transfected cells. When normalized to theuntransfected control cells, the viability of the transfected cells was90%. The mRNA-transfected cells exhibited a reduced rate of celldivision for one day post-transfection, but they regained the normalcell division rate subsequently. The transfected NK cells maintainedapproximately half of the cell number relative to control untransfectedcells 4 days post-transfection (FIG. 1E).

Example 3 Electroloading of Expanded NK Cells with mRNA Coding for aChimeric Receptor

An mRNA coding for an anti-CD19 chimeric receptor was loaded into NKcells by electroloading. As shown in FIG. 2A, 65% of all the viablecells expressed the anti-CD19 chimeric receptor as analyzed by flowcytometry one day post-transfection. The expression of the anti-CD19chimeric receptor increased with the increase of mRNA concentration usedin the electroloading. Using an mRNA concentration of 200 μg/ml,expression of anti-CD19 chimeric receptor was observed for 4 daysfollowing electroloading (FIG. 2B). Recovery of the anti-CD19 chimericreceptor-modified NK cells was similar to that of cellselectrotransfected with an mRNA coding for the marker gene eGFP.

Example 4 Electroloading of Unstimulated Resting NK Cells

Freshly collected resting NK cells were isolated from a peripheral bloodmononuclear cells (PBMC) population using immunoabsorbtion to magneticbeads (Miltenyi Biotec, CD56+ NK Cell Isolation Kit). NK cells werenegatively selected attaining >90% purity with representation by minimalCD3+ cells (FIG. 3A). Electroloading of mRNA encoding for anti-CD19chimeric receptor resulted in expression of anti-CD19 chimeric receptorin between 50 and 60% of electro-loaded resting NK cells (FIG. 3B) 1 daypost-transfection. No significant decrease in the expression ofanti-CD19 chimeric receptor was observed in the electrotransfected NKcells 2 days post-transfection from 1 day post-transfection (FIG. 3C).The viability of un-electroporated control cells and electrotransfectedNK cells at 1 day post-transfection was 75% and 60% respectively, andwas about 80% for transfected cells when normalized againstun-electroporated control cells (FIG. 3D). GFP expression of resting NKcells (>80%) and expanded NK cells (˜80%, see Example 2 above) whenelectrotransfected with mRNA encoding for eGFP was much more efficientcompared to the GFP expression of expanded NK cells (˜50%)electrotransfected with DNA plasmid encoding eGFP.

Example 5 Killing of Leukemia Cells by NK Cells Electrotransfected withmRNA Encoding a Chimeric Receptor

To assay specific killing of leukemia cells by NK cellselectrotransfected by mRNA coding for a chimeric anti-CD19 receptor thecell line OP-1 was used as a target cell. Lysis was carried out in thepresence or absence of CD3+ cells. Depletion of CD3+ cells in theexpanded NK cell population using Dynal beads conjugated with anti-CD3antibody (Invitrogen, Carlsbad, Calif.) designed for removal of CD3+cells by positive selection following the manufacture's protocol wascarried out immediately prior to electroloading with the mRNA encodingfor the chimeric anti-CD19 receptor (FIG. 4A). FIG. 4B shows the similarkilling curve derived from the samples either with or without CD3+ celldepletion.

The specificity of anti-CD19 chimeric receptor for killing leukemiccells was confirmed by comparing killing by NK cells electrotransfectedusing mRNA encoding for the chimeric anti-CD19 receptor to otherwiseidentical NK cells electrotranfected using mRNA encoding for the markergene eGFP or cells that received the same electroporation treatmentabsent any exogenous mRNA. NK cells electroporated in the absence ofexogenous mRNA, NK cells electrotransfected using mRNA coding for eGFPand NK cells electrotransfected using mRNA coding for the chimericanti-CD19 receptor exhibited similar cell viability followingelectroloading (FIG. 5A). NK cells electroporated in the absence ofexogenous mRNA, and NK cells electrotransfected using mRNA coding foreGFP both failed to kill calcein labeled OP-1 leukemia cells (FIGS. 5Band 5C). NK cells electrotransfected using mRNA encoding for thechimeric anti-CD19 receptor when mixed with calcein labeled OP-1 cellsresulted in significant lysis of the target labeled OP-1 cells (FIGS. 5Band 5C).

Specific lysis of labeled OP-1 cells by NK cells electrotransfectedusing mRNA encoding for the chimeric anti-CD19 receptor was proportionalto the concentration of the mRNA encoding for the chimeric anti-CD19receptor. Cell killing was seen as early as 3 hours post transfection(FIG. 6A). Four hours of co-culture resulted in approximately 80% lysisof the OP-1 cells at a ratio of 1:2 (effector:target (E:T)). Overnightco-culture resulted in almost 100% lysis of the OP-1 cells at the same1:2 ratio. Anti-CD19 chimeric receptor-transfected cells maintainedtheir specific killing activity for 3 days post transfection, as shownin FIGS. 6A-6D. On day 3 post transfection, anti-CD19 chimericreceptor-transfected cells lysed 80% and 100% of the target OP-1 cellsafter 4 hours of co-culture at 2:1 and 4:1 E:T ratio respectively (FIG.6D). FIG. 7A summarizes cytotoxicity results of primary NK cells derivedfrom two different donors. Anti-CD19 chimeric receptorelectrotransfected NK cells from both donors showed similar target celllysis efficiency and kinetics.

Specific killing of target cell line OP-1 by resting NK cells 1 dayafter electroloading was assayed. As shown in FIG. 7B, the anti-CD19chimeric receptor-electro-loaded resting NK cells could efficiently killOP-1 cells. At an E:T ratio of 8:1, about 80% of the target cells werelysed in the 4 hour co-cultivation killing assay. The naïve andGFP-expressing resting NK cells did not lyse target OP-1 cells. RestingNK cells from both donors demonstrated similar lysis activity.

Example 6 Resting NK Cells Electrotransfected Using mRNA Encoding forthe Chimeric Anti-CD19 Showed Specific Killing of Allogeneic PrimaryB-Lineage Leukemia Cells

NK cells were electrotransfected using a mRNA encoding for the chimericanti-CD19 receptor, mRNA encoding for eGFP, and NK cells electroporatedin the absence of exogenous mRNA were assayed for their ability tospecifically lyse labeled B-CLL target cells. A significant percentageof B-CLL cells were lysed by NK cells electrotransfected using mRNAencoding for the chimeric anti-CD19 receptor as compared to NK cellselectrotransfected using an mRNA encoding for eGFP or NK cellselectroporated in the absence of exogenous mRNA. Target B-CLL from twodonors were used in these assays and the results were summarized in FIG.8A. Resting NK cells electrotransfected using a mRNA encoding for thechimeric CD19 receptor could kill B-CLL cells at least for two daysafter transfection (FIG. 8B) and the killing of these labeled B-CLLcells was significantly higher than by either the NK cellselectrotransfected using mRNA encoding for eGFP or NK cellselectroporated in the absence of exogenous mRNA.

Example 7 DNA Uptake is Toxic to Resting PBLs

Resting PBLs were electroporated in the presence of plasmid DNA encodingfor eGFP under the control of a CMV promoter, mRNA encoding for theeGFP, and macromolecule FITC-dextran (500 kD). The viability and theexpression level were monitored by trypan blue exclusion and flowcytometry analysis for up to 7 days post transfection. FIG. 9A showstypical FACS analysis data. As shown in FIGS. 9B and 9C, the viabilityof cells electroporated in the presence of DNA was much lower incomparison to cells electroporated with mRNA or FITC-dex. In addition,GFP expression was much lower in cells transfected with the DNA-GFP ascompared to the mRNA-GFP (FIG. 9D).

DNA uptake also resulted in enhanced apoptosis in resting PBLs. RestingPBLs were transfected with 200 ug/ml of plasmid DNA encoding for DsRedunder the control of a CMV promoter and analyzed 1 day posttransfection. The transfected cells were labeled with apoptosisindicator FITC-VAD-FMK (Promega, Madison, Wis.) following the productinstructions. Apoptosis and transgene expression of the transfectedcells were analyzed by FACS (FIG. 10A). The percentage of apoptoticcells was more than twice as high in the transfected cells as comparedto control cells (FIG. 10A). The transfected cells withoutelectroporation, DNA or caspase inhibitor (Enzyme System Product,Livermore, Calif.) (−E−D−I), with electroporation but without DNA orcaspase inhibitor (+E−D−I), with electroporation and caspase inhibitorbut without DNA (+E−D+I), with electroporation and DNA but withoutcaspase inhibitor (+E+D−I), and with electroporation, DNA and caspaseinhibitor (+E+D+I) were analyzed with Cell Death Detection ELISAPLUS kit(Roche, Indianapolis, Ind.) (FIG. 10B). Caspase inhibitor could onlyslow the cell death of the DNA-transfected resting PBL, not stop it.

Example 8 Chimeric Receptor Expression in Resting PBMCs, NK Cells, PBLsfrom Healthy Donors and Resting PBMC and CD3+ T Cells from CLL Patient

αCD19 chimeric expression was evaluated in resting PBMCs (FIG. 11A),resting NK cells (FIG. 11B), resting PBLs (FIG. 11C) from healthy donorsand resting PBMC (FIG. 11D) and CD3+ cells (FIG. 11E) from CLL patient.FIG. 12A shows high viability of PBLs transfected with an mRNA encodinga αCD19 chimeric receptor for at least 7 days post transfection. FIG.12B shows dependence of viable cell recovery on time post transfection.FIG. 12C shows expansion of transfected PBL analyzed with CFSE. FIG. 12Dshows expansion of CD3+ cells analyzed with CFSE.

Example 9 Specificity of Allogenic Target Cell/Cell Line Killing byαCD19 Chimeric Receptor Transfected Resting PBLs

In this study, the specificity of allogeneic target cell/cell linekilling by αCD19 chimeric receptor transfected resting PBLs wasevaluated. FIG. 13A shows typical FACS analysis result of OP-1 cell linekilling. FIG. 13B shows specific OP-1 killing by 2 donors of transfectedPBLs, whereas naïve or GFP-transfected PBLs did not specifically killOP-1 cells. FIG. 13C shows non-specific K562 cell killing by transfectedPBLs. FIG. 13D shows specific CLL cell killing.

Example 10 Specificity of Autologous B or Purified CD19+ CLL CellKilling by αCD19 Chimeric Receptor Transfected Resting NK Cells, RestingPBMCs, Resting PBLs from Healthy Donors and Resting PBMC and CD3+ Cellsfrom CLL Patient

FIGS. 14A-14D show specific autologous B or purified CD19+ CLL cellkilling by resting NK cells (1d post transfection) (FIG. 14A); restingPBMCs (3d post transfection) (FIG. 14B); resting PBLs (1d posttransfection) (FIG. 14C); resting PBMC from CLL patient (FIG. 14D) andresting CD3+ T cells from CLL patient (FIG. 14E) after transfection withαCD19 chimeric receptor. FIGS. 15A-15D show the duration of specifickilling of autologous B cells by transfected resting PBLs with αCD19chimeric receptor. The four-hour killing assay was performed at 1 daypost transfection (FIG. 15A), 2 days post transfection (FIG. 15B), 3days post transfection (FIG. 15C), and 7 days post transfection (FIG.15D).

Materials and Methods

Cells.

The CD19+ human B-lineage ALL cell line, OP-1 (developed at St. JudeChildren's Research Hospital), and the genetically engineered myeloidleukemia cell line, K562, co-expressing 4 1BB ligand and membrane boundIL-15 (K562-4-15), also developed at St. Jude Children's ResearchHospital, were maintained in RPMI-1640 supplemented with 10% fetalbovine serum and antibiotics. Primary leukemic cells from two patientswith B-CLL were obtained by directly collecting the cells in theinterfacial layer after Ficoll plaque density gradient centrifugation,frozen after two rounds of PBS wash until use. Primary peripheral bloodmononuclear cells (PBMC) from healthy donors were prepared fromleukapheresis product purchased from BRT Laboratories, Inc. (Baltimore,Md.). PBMCs were obtained directly from the interfacial layer instandard Ficoll density gradient centrifugation, washed twice withphosphate buffered saline (PBS), and frozen and stored in liquidnitrogen until use. The primary cells, whenever used, were cultured inRPMI-1640 supplemented with 10% fetal bovine serum and antibiotics. CD3+cells were obtained by negative purification using Miltenyi kit.

The resting NK cells were negatively selected by following the protocolsupplied with the Miltenyi kit (Auburn, Calif.) and frozen in liquidnitrogen until use. Primary NK cells were expanded as previouslydescribed by Imai et al. Peripheral blood mononuclear cells werecultured with thawed K562 cells that express 4-1BB ligand andmembrane-bound IL-15 (K562-4-15) provided by St. Jude Children'sResearch Hospital and which were irradiated with 10,000 to 20,000 radprior to culturing with NK cells. Culturing of the NK cells with thetarget cells to allow for NK cell killing was performed in the presenceof 10 IU/ml-100 U/ml IL-2, 10% FBS and antibiotics.

PBMCs were prepared by incubating the thawed PBMCs in a centrifuge tubefor 30 minutes after thawing and collecting all cells by centrifugation.PBLs were prepared by culturing the thawed PBMC in tissue culture flaskfor 1-2 hours and only collecting the suspended cells.

Molecules for Electroloading.

The cloning of anti-CD19 chimeric receptor into pVAX1 (Invitrogen,Carlsbad, Calif.) vector was performed by digesting the parent plasmidpMSCVanti-CD19BBZ encoding a single chain antibody conjugated with the4-1 BB intercellular domain and the CD3ζ domain (generated at St. JudeChildren's Research Hospital) and the pVAX1 vector with EcoR I and Xho Iand ligated using T4 DNA ligase. mRNA encoding for anti-CD19 chimericreceptor was in vitro transcribed with T7 polymerase using an AmbionmMESSAGE mMACHINE T7 Ultra kit (Ambion, Austin, Tex.) with the clonedtemplate of the pVAX1 vector containing anti-CD19 chimeric receptor.mRNA quality and quantity was analyzed by 1% agarose gel after 15minutes denaturation at 70° C. in mRNA denaturation buffer (Invitrogen,Carlsbad, Calif.) and OD260/280 measurement. The plasmid DNA encodingfor eGFP on the pCI (Promega, Madison, Wis.) backbone under CMV promoterwas used for DNA transfection. The mRNA encoding for GFP was producedusing the pCI-eGFP and the same Ambion kit as mentioned above.FITC-dextran was purchased from Sigma (St. Luis, Mo.). TheFITC-conjugated control siRNA was purchased from Qiagen (Valencia,Calif.).

Transfection.

The resting NK cells in frozen medium (10% DMSO in FBS) were thawed in37° C. water bath, incubated for 0.5-1 h at 37° C. in the prewarmedfresh full medium (RPMI-1640+10% FBS+ antibiotics) with volume of 10×that of frozen medium and ready for transfection. The expanded NK cellswere harvested at the indicated time points for transfection. Beforetransfection, the expanded NK cells were washed with MXCT EP bufferonce. The unstimulated resting cells were washed 2× in PBS containing0.5% FBS and 2 mM EDTA and 3× in MXCT EP buffer containing additionally0.1% BSA. After washing, expanded NK and resting NK, PBL, PBMC. T, andCD8+ cells were suspended in MXCT EP buffer, mixed with molecules to beloaded/transfected, transferred into MXCT chamber, transfected withprogram “Expanded-NK Cell #3” and “Resting-NK #1” for expanded andresting NK cells respectively in MXCT GT system (Maxcyte, Gaithersburg,Md.), transferred into incubation tube, incubated for 20 minutes at 37°C., and returned to the culture medium. The loading or expressionefficiency was analyzed by flow cytometry.

Detection of the Expression of Chimeric Receptor and Immunophenotyping.

The transfected NK cells were stained with goat anti-mouse (Fab)2polyclonal antibody conjugated with biotin (Jackson immuno Researchlabs, West Grove, Pa.) followed by peridinin chlorophyll protein—(PerCp;Becton Dickinson, San Jose, Calif.) labeled streptavidin staining. Thepositive cells was gated according to the background cells with goatbiotin-conjugated IgG followed by streptavidin-PerCp.

The following antibodies were used for immunophenotypic characterizationof expanded and transfected NK cells: anti-CD3 conjugated withfluorescein isothiocyanate (FITC), anti-CD19 conjugated withphycoerythrin (PE), anti-CD16-PE, and anti-CD56-PE. Antibody stainingwas analyzed by a FACSCalibur (Becton Dickinson).

Cell Killing Assays.

To facilitate the large number of cell killing studies, a cell killingassay was developed based on acetoxmethyl-calcein (calcein-AM, MolecularProbes, Eugene, Oreg.) staining and flow cytometry. Briefly, calcein-AMpre-labeled target cells (100 μl) were co-cultured with 100 μl of eithertransfected, non-transfected primary NK cells or just fresh medium atvarious effector to target (E:T) ratios in each well of a 96-wellU-bottom tissue culture plate (Costar, Cambridge, Mass.). The 96-wellplate was centrifuged at 400 g for 5 minutes prior to cell culture at a37° C., 5% CO₂ incubator. The cells were resuspended in the originalculture media, transferred to FACS tubes for FACS analysis at indicatedtime points.

In some studies, cell killing assays described in Imai, et al. werefollowed. Briefly, plain target cells 10⁵ cells in 100 μl wereco-cultured with 100 μl of either transfected, non-transfected primaryNK cells or just fresh medium at various effector to target (E:T) ratiosin each well of a 96-well U-bottom tissue culture plate (Costar,Cambridge, Mass.). After 400 g×5 minutes centrifugation, the cells werecultured for desired cell-killing time. The cells were harvested andco-stained with anti-CD19-FITC and anti-CD56-PE antibodies for 20minutes on ice. After washing in PBS, the cells were resuspended with200 μl of PBS and analyzed by flowcytometry.

The cultures were performed in the absence of exogenous IL-2. FACSanalysis was performed using a FACSCalibur with 15 second collection.The specific cell lysis rate (%) was calculated by100−N_(target)/N_(control)×100, where N_(target) is the number of viabletarget cells co-cultured with NK cells and the N_(control) is the numberof viable target cells cultured alone.

Example 11 CAR-Expressing PBMCs Kill HS-Sultan Cells In Vitro and InVivo

Human PBMCs were electroloaded with mRNA encoding anti-CD19-BBz. Four orseven days post transfection, PBMCs (transfected or non-transfected(Naïve)) were mixed in vitro with calceinAM-prelabeled HS-Sultan cells,a leukemia cell line that is CD19+, at various effector:target (E:T)ratios. Cell cytotoxicity was performed by FACS 4 hours after mixing. Asshown in FIG. 16, the transfected PBMCs were able to kill HS-Sultancells at 4 days and 7 days at all E:T ratios tested, with the killingbeing more effective at 4 days than at 7 days. In addition, increasedkilling was achieved with higher E:T ratios.

To demonstrate that CAR-expressing PBMCs could kill tumor cells in vivo,a HS-Sultan subcutaneous co-mixing model in Beige SCID mice was used.PBMC 1 day post-mRNA transfection (transfected or non-transfected) withmRNA encoding anti-CD19-BBz were mixed with HS-Sultan cells at differentratios and subcutaneously injected into mice (5 mice/group) as indicatedin Table 1.

TABLE 1 Study PBMC- group purpose E:T Sultan # PBMC# CAR# Mouse # 1control 0 1e6 0 0 5 2 control  20:1 1e6 2e7 0 5 3 treatment  20:1 1e6 0 2e7 5 4 6.7:1 1e6 0 6.7e6 5 5 2.3:1 1e6 0 2.3e6 5 6 0.7:1 1e6 0 0.7e6 5

Tumor volume in the mice was measured at day 0, 14, 18, and 26. As shownin FIG. 17, no measurable tumor developed in study groups 3, 4, and 5.Measurable tumor similar to that in the PBMC control was found in thestudy group receiving the lowest dose of CAR-expressing PBMCs (0.7e6).

In a further in vitro study, cytokine-induced NK cells (LAK) weretransfected with mRNA encoding anti-CD19-BBz and mixed with HS-Sultancells for a cytotoxicity study. LAK cells are NK cells that have beenstimulated to be cytotoxic to tumor cells by Interleukin-2. As shown inFIG. 18, while control LAK cells (conventional) were cytotoxic againstHS-Sultan cells, the transfected LAK cells (Engineered LAK cells) weresignificantly more cytotoxic.

Example 12 Effect of Intracellular Domains on CAR Expression

The effect of intracellular domains in the chimeric antigen receptor wasevaluated with the following four anti-mesothelin CARs: ss1-28-BBz,ss1-28z, ss1-BBz, or ss1-z. RNA was prepared that encodes CAR composedof an anti-mesothelin (ss1) murine single-chain Fv binding domain withthe combination of 3 intracellular activation domains derived from 41BBand CD28, and the cytoplasmic portion of the TcRz□ chain.

Expanded T cells were electroloaded with ss1-myc-28-BBz, ss1-28z,ss1-BBz, or ss1-z mRNAs. As shown in FIGS. 19 and 20, CAR expressiondecreased more quickly in the T cells transfected with mRNAs containingthe CD28 intracellular domain (ss1-myc-28-BBz and ss1-28z). The decreasein CAR expression correlated with a decrease in the ability of the Tcells to kill cancer cells. As shown in FIG. 21, only ss1-BBz and ss1-z(i.e., the mRNAs that did not have the CD28 intracellular domain)transfected T cells maintained K562 cell killing ability 3 days posttransfection.

The expression of ss1-28z was compared in PBMCs and expanded T cells. Asshown in FIG. 22, ss1-28z expression decreases faster in T cells than inPBMCs. This may be a result of the T cells doubling faster than thePBMCs. The ss1-28z transfected PBMCs maintained K562+ cell killingability at 4 days post transfection (FIG. 23).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for preparing transiently modified unstimulated restingperipheral blood mononuclear cells (PBMCs), the method comprising: (a)obtaining freshly collected unstimulated resting PBMCs from a peripheralblood sample; and (b) electroloading the PBMCs with an mRNA encoding fora chimeric receptor to generate electroloaded PBMCs expressing thechimeric receptor on their surface; wherein the electroloading comprisesflow electroporation.
 2. The method of claim 1, wherein the PBMCs areelectroloaded less than 5 hours from the time the PBMCs were originallyobtained from a subject.
 3. The method of claim 1, wherein the PBMCs donot undergo cell division prior to (b).
 4. The method of claim 1,wherein the chimeric receptor is an anti-CD19 chimeric receptor.
 5. Themethod of claim 1, wherein the chimeric receptor is an anti-CD20,anti-FBP, anti-TAG-72, anti-CEA, anti-carboxyanhydrase IX, anti-CD171,anti-IL-13 receptor, anti-G(D)2, anti-PSMA, anti-mesothelin,anti-Lewis-Y, or anti-CD30 chimeric receptor.
 6. The method of claim 1,wherein the chimeric receptor comprises a CD28 intracellular domain. 7.The method of claim 1, wherein the chimeric receptor does not comprise aCD28 intracellular domain.
 8. A method for administering cells to asubject, the method comprising: (a) obtaining freshly collectedunstimulated resting PBMCs from a peripheral blood sample; (b)electroloading the PBMCs with an mRNA encoding for a chimeric receptorto generate electroloaded PBMCs expressing the chimeric receptor ontheir surface; and (c) administering the electroloaded PBMCs to thesubject, wherein the electroloading comprises flow electroporation. 9.The method of claim 8, wherein the PBMCs are electroloaded less than 5hours from the time the PBMCs were originally obtained from a subject.10. The method of claim 8, wherein the PBMCs do not undergo celldivision prior to (b).
 11. The method of claim 8, wherein the chimericreceptor is an anti-CD19 chimeric receptor.
 12. The method of claim 8,wherein the chimeric receptor is an anti-CD20, anti-FBP, anti-TAG-72,anti-CEA, anti-carboxyanhydrase IX, nati-CD171, anti-IL-13 receptor,anti-G(D)2, anti-PSMA, anti-mesothelin, anti-Lewis-Y, or anti-CD30chimeric receptor.
 13. The method of claim 8, wherein the chimericreceptor comprises a CD28 intracellular domain.
 14. The method of claim8, wherein the chimeric receptor does not comprise a CD28 intracellulardomain.
 15. The method of claim 8, wherein the PBMCs are autologous tothe subject.
 16. The method of claim 8, wherein the PBMCs are allogenicto the subject.
 17. The method of claim 8, wherein (a) comprisescollecting the peripheral blood sample from a donor and separating thePBMCs in the sample from the non-PBMCs in the sample.
 18. The method ofclaim 8, wherein the method comprises treating or preventing a cancer inthe subject.
 19. The method of claim 18, wherein the cancer is breastcancer, lung cancer, prostate cancer, ovarian cancer, brain cancer,liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer,head & neck cancer, bone cancer, esophageal cancer, bladder cancer,uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer,testicular cancer, or leukemia.
 20. The method of claim 19, wherein theleukemia is acute lymphocytic leukemia (ALL), acute myelogenous leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CML), or mantle cell lymphoma (MCL).
 21. The method of claim 8, whereinthe electroloaded PBMCs are administered to the subject by intravenousinjection.
 22. The method of claim 8, wherein the electroloaded PBMCsare administered to the subject by intratumoral injection.